The overall goal of this project is to characterize the effects of tetracyclines on osteoclast and osteoblast function in vitro. Preliminary data indicate that tetracyclines inhibit osteoclastic activity while stimulating osteoblastic function. The inhibitory effects of tetracyclines will be examined by studying key aspects of osteoclastic function: a) osteoclast ruffled border development will be evaluated using cultured fetal rat long bones and morphometric techniques; b) resorption of devitalized bone by isolated osteoclasts will be assessed with disaggregated rat osteoclasts cultured on devitalized bone and an enriched preparation of avian osteoclasts cultured on prelabeled bone chips; c) acidification mechanism of osteoclasts will be assessed with acridine orange fluorescence in whole cells and in plasma membranes; d) activities of osteoclastic lysosomal cysteine proteinases will be accomplished by spectrofluorometric assay of cathepsin B and L; e) osteoclast cytosolic free calcium will be examined by single cell measurements of cytosolic calcium using Fura-2; f) osteoclast podosome formation will be assessed in chicken osteoclasts by rhodamine-labelled phalloidin and, g)) osteoclast oxygen radical production will be examined in stimulated avian osteoclasts by established spectrophotometric assays. The anabolic effects of tetracyclines will be evaluated using isolated rodent periosteal and endosteal osteoprogenitor cells and osteoblasts. The studies will include tetracyclines effects on a) parameters of osteoblastic function such as proliferation, collagen synthesis, collagen types, collagenase production, alkaline phosphatase action, proteoglycan synthesis, osteocalcin production, bone nodule formation, and interaction with known immune and bone regulatory factors; and b) to determine the mechanism of action of tetracycline on eliciting a biological effect by using second messenger assays. Furthermore, molecular biological technology will be used to determine whether the effects of tetracyclines are mediated through translation or transcription control. Results from these studies should provide a rational basis for an understanding of the effects of tetracycline in vivo and their potential therapeutic value in the management of bone destructive disorders.